The present invention relates to methods for the analysis of samples by mass spectrometry. The apparatus and methods for ion transport and analysis described herein are enhancements of the techniques referred to in the literature relating to mass spectrometry—an important tool in the analysis of a wide range of chemical compounds. Specifically, mass spectrometers can be used to determine the molecular weight of sample compounds. The analysis of samples by mass spectrometry consists of three main steps—formation of gas phase ions from sample material, mass analysis of the ions to separate the ions from one another according to ion mass, and detection of the ions. A variety of means and methods exist in the field of mass spectrometry to perform each of these three functions. The particular combination of the means and methods used in a given mass spectrometer determine the characteristics of that instrument.
To mass analyze ions, for example, one might use magnetic (B) or electrostatic (E) analysis, wherein ions passing through a magnetic or electrostatic field will follow a curved path. In a magnetic field, the curvature of the path will be indicative of the momentum-to-charge ratio of the ion. In an electrostatic field, the curvature of the path will be indicative of the energy-to-charge ratio of the ion. If magnetic and electrostatic analyzers are used consecutively, then both the momentum-to-charge and energy-to-charge ratios of the ions will be known and the mass of the ion will thereby be determined. Other mass analyzers are the quadrupole (Q), the ion cyclotron resonance (ICR), the time-of-flight (TOF), the Orbitrap, and the quadrupole ion trap analyzers. The analyzer used in conjunction with the method described here may be any of a variety of these.
Before mass analysis can begin, gas phase ions must be formed from a sample material. If the sample material is sufficiently volatile, ions may be formed by electron ionization (EI) or chemical ionization (CI) of the gas phase sample molecules. Alternatively, for solid samples (e.g., semiconductors, or crystallized materials), ions can be formed by desorption and ionization of sample molecules by bombardment with high energy particles. Further, Secondary Ion Mass Spectrometry (SIMS), for example, uses keV ions to desorb and ionize sample material. In the SIMS process a large amount of energy is deposited in the analyte molecules, resulting in the fragmentation of fragile molecules. This fragmentation is undesirable in that information regarding the original composition of the sample (e.g., the molecular weight of sample molecules) will be lost.
For more labile, fragile molecules, other ionization methods now exist. The plasma desorption (PD) technique was introduced by Macfarlane et al. (D. F. Torgerson, R. P. Skowronski, and R. D. Macfarlane, Biochem. Biophys. Res Commoun. 60 (1974) 616)(“McFarlane”). Macfarlane discovered that the impact of high energy (MeV) ions on a surface, like SIMS would cause desorption and ionization of small analyte molecules. However, unlike SIMS, the PD process also results in the desorption of larger, more labile species (e.g., insulin and other protein molecules).
Additionally, lasers have been used in a similar manner to induce desorption of biological or other labile molecules. See, for example, Cotter et al. (R. B. VanBreeman, M. Snow, R. J. Cotter, Int. J. Mass Spectrom. Ion Phys. 49 (1983) 35; Tabet, J. C.; Cotter, R. J., Tabet, J. C., Anal. Chem. 56 (1984) 1662; or R. J. Cotter et al., Anal. Instrument. 16 (1987) 93). Cotter modified a CVC 2000 time-of-flight mass spectrometer for infrared laser desorption of non-volatile biomolecules, using a Tachisto (Needham, Mass.) model 215G pulsed carbon dioxide laser. The plasma or laser desorption and ionization of labile molecules relies on the deposition of little or no energy in the analyte molecules of interest.
The use of lasers to desorb and ionize labile molecules intact was enhanced by the introduction of matrix assisted laser desorption ionization (MALDI) (K. Tanaka, H. Waki, Y. Ido, S. Akita, Y. Yoshida, T. Yoshica, Rapid Commun. Mass Spectrom. 2 (1988) 151 and M. Karas, F. Hillenkamp, Anal. Chem. 60 (1988) 2299). In the MALDI process, an analyte is dissolved in a solid, organic matrix. Laser light of a wavelength that is absorbed by the solid matrix but not by the analyte is used to excite the sample. Thus, the matrix is excited directly by the laser, and the excited matrix sublimes into the gas phase carrying with it the analyte molecules. The analyte molecules are then ionized by proton, electron, or cation transfer from the matrix molecules to the analyte molecules. This process (i.e., MALDI) is typically used in conjunction with time-of-flight mass spectrometry (TOFMS) and can be used to measure the molecular weights of proteins in excess of 100,000 Daltons.
Further, Atmospheric Pressure Ionization (API) includes a number of ion production means and methods. Typically, analyte ions are produced from liquid solution at atmospheric pressure. One of the more widely used methods, known as electrospray ionization (ESI), was first suggested by Dole et al. (M. Dole, L. L. Mack, R. L. Hines, R. C. Mobley, L. D. Ferguson, M. B. Alice, J. Chem. Phys. 49, 2240, 1968). In the electrospray technique, analyte is dissolved in a liquid solution and sprayed from a needle. The spray is induced by the application of a potential difference between the needle and a counter electrode. The spray results in the formation of fine, charged droplets of solution containing analyte molecules. In the gas phase, the solvent evaporates leaving behind charged, gas phase, analyte ions. This method allows for very large ions to be formed. Ions as large as 1 MDa have been detected by ESI in conjunction with mass spectrometry (ESMS).
In addition to ESI, many other ion production methods might be used at atmospheric or elevated pressure. For example, MALDI has recently been adapted by Laiko et al. to work at atmospheric pressure (Victor Laiko and Alma Burlingame, “Atmospheric Pressure Matrix Assisted Laser Desorption”, U.S. Pat. No. 5,965,884, and Atmospheric Pressure Matrix Assisted Laser Desorption Ionization, poster #1121, 4th International Symposium on Mass Spectrometry in the Health and Life Sciences, San Francisco, Aug. 25-29, 1998) and by Standing et al. at elevated pressures (Time of Flight Mass Spectrometry of Biomolecules with Orthogonal Injection+Collisional Cooling, poster #1272, 4th International Symposium on Mass Spectrometry in the Health and Life Sciences, San Francisco, Aug. 25-29, 1998; and Orthogonal Injection TOFMS Anal. Chem. 71(13), 452A (1999)). The benefit of adapting ion sources in this manner is that the ion optics (i.e., the electrode structure and operation) in the mass analyzer and mass spectral results obtained are largely independent of the ion production method used.
Many different types of analyzers have been used to mass analyze sample ions. One important type of mass analyzer is the quadrupole mass analyzer. There are also several types of quadrupole analyzers. Among them are the quadrupole filter, the quadrupole trap—a.k.a. the Paul trap—the cylindrical ion trap, linear ion trap, and the rectilinear ion trap.
The conventional quadrupole filter consists of four rods equally spaced at a predetermined radius around a central axis. A radio frequency (RF)—e.g. a 1 MHz sine wave—potential is applied between the rods. The potential on adjacent rods is 180° out of phase. Rods on opposite sides of the quadrupole axis are electrically connected—i.e. the quadrupole is formed as two pairs of rods. The quadrupole has an entrance end and an exit end. Ions to be filtered are injected into the entrance end of the quadrupole. These ions travel along the axis of the quadrupole to the exit end. The RF potential applied between the rods will tend to confine the ions radially. Applying a DC as well as an RF potential between the pairs of rods will cause ions of only a limited mass range to be transmitted through the quadrupole. Ions outside this mass range will be filtered away and will not reach the exit end.
In a quadrupole mass analyzer, ions transmitted through the quadrupole are detected as ion signals via a channeltron detector. To produce a mass spectrum the quadrupole parameters are “scanned” and the ion signals are recorded as a function of the scan parameters. In the so-called “mass-selective stability” mode of operation the amplitudes of RF and DC voltages applied to the quadrupole rods are ramped at a constant RF/DC ratio. At each point in the ramp, ions of nominally a single m/z have a stable trajectory and are transmitted. Recording the ion signal as a function of the ramp thus yields a mass spectrum.
The Paul ion trap (a.k.a. quadrupole ion trap) is based on a similar principle and construction as the quadrupole filter, however, as the name implies, ions are trapped in the Paul trap before they are mass analyzed. Also unlike the quadrupole filter, the Paul trap is cylindrically symmetric. The Paul trap is constructed using three rotationally symmetric hyperbolic electrodes. Two “end cap” electrodes are placed one on either side of a central “ring electrode”. Applying an RF potential between the ring electrode and the end caps forms a quadrupolar pseudopotential well in the interior volume of the trap. In a typical analysis ions enter the trap through apertures in one of the end caps, lose kinetic energy via collisions with gas in the trap and thereby become trapped in the pseudopotential well.
The quadrupole ion trap is typically operated in one of two modes—the mass selective instability mode or the resonance ejection mode. The mass selective instability mode differs from the mass selective stability mode described above in that ions are detected when their trajectories become unstable. Initially, a group of analyte ions is trapped near the center of the quadrupole ion trap. The ions will oscillate about the center of the trap with a frequency related to the m/z of the ion. When performing a mass selective instability scan, the amplitude of the RF potential applied to the ring electrode is ramped to higher values. At each point in the RF ramp, ions below a given m/z have unstable trajectory and are ejected from the trap. The given “cutoff” m/z is a linear function of the RF amplitude. Thus, recording the ion signal as a function of the ramp yields a mass spectrum.
A similar principle is applied when operating in the resonance ejection mode. However, in resonance ejection mode, an additional AC potential is applied between the end cap electrodes. The ions are excited not only by the RF as in selected ion instability mode but also by the supplemental AC. Therefore the ions are ejected more quickly from the trap—i.e. earlier in the ramp. Because ions are ejected from the trap at lower RF amplitudes, experiments using resonance ejection can be used to analyze higher m/z ions than can be achieved in mass selective instability experiments.
Many additional methods of manipulating ions in traps are know from the prior art including ion trapping, precursor isolation, CID, tandem mass spectrometry, ion-ion reactions, etc. Such methods may be applied, not only to the Paul trap as described above, but also to the other prior art trapping devices described below and to the present invention.
The cylindrical ion trap (CIT) is a simplified form of the Paul trap described above. The cylindrical ion trap is formed by a central cylinder instead of a hyperbolic ring electrode, and two flat plates instead of hyperbolic end caps. Due the simplified geometry of these electrodes, the CIT has a lower resolution than conventional Paul traps of similar inner diameter. However, because of its simplified construction—i.e. flat end caps and cylindrical ring electrode instead of hyperbolic surfaces—the CIT can more readily be miniaturized.
Yet another type of ion trap is the “linear ion trap”. In principle, any type of multipole in which ions are trapped may be considered a linear ion trap, however, the device now commonly referred to as a linear ion trap can be used not only to trap ions but also to analyze them. As described by Schwartz et al. (J. C. Schwartz, M. W. Senko, and J. E. P. Syka, J. Am. Soc. Mass Spectrom. 13, 659 (2002)) a linear ion trap includes two pairs of electrodes or rods, which contain ions by utilizing an RF quadrupole trapping field in two dimensions, while a non-quadrupole DC trapping field is used in the third dimension. Simple plate lenses at the ends of a quadrupole structure can provide the DC trapping field. This approach, however, allows ions which enter the region close to the plate lenses to be exposed to substantial fringe fields due to the ending of the RF quadrupole field. These non-linear fringe fields can cause radial or axial excitation which can result in loss of ions. In addition, the fringe fields can cause shifting of the ions frequency of motion in both the radial and axial dimensions.
An improved electrode structure of a linear quadrupole ion trap 11, which is known from the prior art, is shown in FIG. 1. The quadrupole structure includes two pairs of opposing electrodes or rods, the rods having a hyperbolic profile to substantially match the equipotential contours of the quadrupole RF fields desired within the structure. Each of the rods is cut into a main or central section and front and back sections. The two end sections differ in DC potential from the central section to form a “potential well” in the center to constrain ions axially. An aperture or slot 12 allows trapped ions to be selectively resonantly ejected in a direction orthogonal to the axis in response to AC dipolar or quadrupolar electric fields applied to the rod pair containing the slotted electrode. In this figure, as per convention, the rod pairs are aligned with the x and y axes and are therefore denoted as the X and Y rod pairs.
In prior art according to Song et al. (Y. Song, G. Wu, Q. Song, R. G. Cooks and Z. Ouyang, J Am. Soc Mass Spectrom. 17, 631 (2006) and U.S. Pat. No. 6,838,666 which is incorporated herein by reference), the hyperbolic rods of the conventional 2D linear ion trap were replaced by rectangular electrodes. This design (shown in FIG. 2) is now known as a rectilinear ion trap (RIT). According to Song et al. the trapping volume is defined by x and y pairs of spaced flat or plate RF electrodes 15, 16 and 13, 14 in the zx and zy planes. Ions are trapped in the z direction by DC voltages applied to spaced flat or plate end electrodes (not shown) in the xy plane disposed at the ends of the volume defined by the x, y pair of plates, or by DC voltages applied together with RF in sections 18 and 19 each comprising pairs of flat or plate electrodes 15a, 16a and 13a, 13b. In addition to the RF sections flat or plate end electrodes can be added. The ions are trapped in the x, y direction by the quadrupolar RF fields generated by the RF voltages applied to the plates. Ions can be ejected along the z axis through apertures formed in the end electrodes or along the x or y axis through apertures formed in the x or y electrodes. The ion trap is generally operated with the assistance of a buffer gas. Thus, when ions are injected into the ion trap they lose kinetic energy by collision with the buffer gas and are trapped by the DC potential well. While the ions are trapped by the application of RF trapping voltages AC and other waveforms can be applied to the electrodes to facilitate isolation or excitation of ions in a mass selective fashion. To perform an axial ejection scan the RF amplitude is scanned while an AC voltage is applied to the end plates. Axial ejection depends on the same principles that control axial ejection from a linear trap with round rod electrodes (U.S. Pat. No. 6,177,668). In order to perform an orthogonal ion ejection scan, the RF amplitude is scanned and the AC voltage is applied on the set of electrodes which include an aperture. The AC amplitude can be scanned to facilitate ejection. Circuits for applying and controlling the RF, AC and DC voltages are well known.
The addition of the two end RF sections 18 and 19 to the RIT also helps to generate a uniform RF field for the center section. The DC voltages applied on the three sections establish the DC trapping potential and the ions are trapped in the center section, where various processes are performed on the ions in the center section.
The most significant advantage of the RIT over the LIT is that of fabrication. The electrodes composing the RIT, being flat surfaces, are much easier to produce, with precision, than the hyperbolic surfaces of the LIT. As a result, the RIT can be more readily miniaturized than the LIT and can be more readily incorporated into portable instruments.
In order to determine the structure of an original analyte molecule it is often helpful to dissociate molecular ions into fragments. Typically, the fragment ions are then mass analyzed. The masses and mass differences between the fragment ions can be used then to determine the original molecule's structure. There are many means of fragmenting precursor analyte ions—collision induced dissociation (CID), infrared multi photon dissociation (IRMPD), surface induced dissociation (SID), etc. The production of identifiable fragment ions is an important measure of the success of a dissociation method.
Collision induced dissociation (CID) is a widely used prior art method used in tandem mass spectrometry experiments. During CID, the internal energy of precursor ions is increased via collisions between the precursor ion and collision gas. The increased internal energy of the ion causes it to dissociate into one or more fragment ions. Collisional activation of precursor ions is achieved by accelerating the ion via an electric field. In instruments using quadrupole filters, the accelerating electric field is typically applied between adjacent multipoles. That is, analyte ions enter the quadrupole filter. In the filter, ions of the mass of interest—i.e. precursor ions—are selected. The selected precursor ions exit the quadrupole filter and are accelerated by an electric field into a collision cell. The collision cell includes another RF multipole used to confine the ions as they undergo activation and fragmentation. The resulting precursor and fragment ions pass through and out of the collision cell multipole and to downstream optics and/or detectors.
In a multipole trap, activation toward dissociation may be accomplished by resonant excitation of the precursor. In a resonant excitation experiment, the electric field used to accelerate the ions is an RF potential applied between the trapping electrodes at the secular frequency of the precursor. In a conventional Paul trap the excitation electric field, for example, might take the form of a 150 mVp-p sine wave applied between the endcap electrodes for a period of tens of milliseconds. Alternatively, a higher amplitude electric field (˜1 Vpp) might be applied for a shorter time (˜2 ms). Further, as described in the prior art of Glish et al. (C. Cunningham Jr., G. L. Glish, and D. J. Burinsky, J Am Soc Mass Spectrom 17, 81 (2006)) and Schwartz et al. in U.S. Pat. No. 7,102,129, the amplitude of the RF potential confining the ions in the trap may be reduced after collisional activation so that fragment ions of low m/z can be observed.
Another fragmentation method used in tandem mass spectrometry experiments is electron capture dissociation (ECD). The prior art method of ECD (K. Vekey, A. G. Brenton, et al., Int J Mass Spectrom Ion Process 70, 277 (1986); F. W. McLafferty, Mass Spectrometry in the Analysis of Large Molecules, C. J. McNeal, Ed., John Wiley, New York, 1986, pp 107-120; and N. C. Polfer et al., Rapid Commun Mass Spectrom 16, 936 (2002)) activates multiply charged positive precursor ions toward fragmentation by partial neutralization of the ion using low kinetic energy electrons. The energy associated with neutralization is often sufficient to produce prompt fragmentation.
Electron transfer dissociation (ETD) and electron capture dissociation (ECD) tandem mass spectrometry techniques have been shown to be useful for the characterization of peptides and proteins (e.g. top-down analysis). Both techniques produce c- and z-type fragment ions, which are complementary to the b- and y-type fragment ions produced in collision induced dissociation (CID). Additionally ETD and ECD provide more extensive fragmentation than CID, resulting in richer product ion spectra and better sequence coverage. Moreover, ETD and ECD are processes which tend to preserve weakly bound post-translational modifications (PTMs) thereby enabling a means of identification and localization of PTMs by mass spectrometry. Neutral loss scans (in a triple quadrupole or ion trap) in conjunction with CID can be used to look for the loss of PTMs, however, this scanning method is an indirect measurement and not always efficient at identifying all PTMs. The reason why ETD and ECD preserve PTMs is highly debated, and whether the processes are ergodic or non-ergodic does not change the utility of the techniques. The combination of the complementary information to CID, richer sequence coverage, and the identification of PTMs make ETD and ECD powerful analytical proteomics tools.
Prior art instruments primarily combine ETD with conventional Paul ion traps (3-D ion traps), linear ion traps (2-D ion traps), and hybrid quadrupole time of flight mass analyzers (qTOF). For trap analyzers, which have a fixed line width across the mass range, it is necessary to perform charge manipulation techniques to reduce the charge of the ions if complex ion populations are to be resolved. Reducing the number of charges on an ion results in a larger spacing between the isotopes and also shifts the ion m/z to a region of the mass spectrum that allows the isotopes to be resolved and the actual charge state and molecular mass determined.
In performing ETD experiments in a 2-D or 3-D ion trap, the spatial overlap between reagent and analyte ions is inherent to the operation of the device. Because the pressure is relatively high, both positive and negative ions are collisionally cooled to the center of the storage device. As a result the reagent and analyte ions occupy nearly the same volume. This strong spatial overlap, of course, tends to promote the ETD reaction. This spatial overlap between the reagent and analyte ions can be optimized but does not change from experiment to experiment. The efficiency of ETD in the 3-D traps suggests that it may be possible to generate ETD data without the need to average multiple mass spectra. In addition the time necessary for the accumulation and reaction for an ETD experiment are typically amenable to on-line separations.
Xia et al. demonstrated an experimental setup in which ions were trapped in a linear quadrupole ion trap using only RF potentials (Xia, Y.; Chrisman, P. A.; Erickson, D. E.; Liu, J.; Liang, X. R.; Londry, F. A.; Yang, M. J.; McLuckey, S. A. Anal. Chem. 2006, 78, 4146-54). Once trapped, the analyte ions were reacted with ETD reagent ions. Product ions and remaining analyte ions were transferred from the quadrupole trap to an orthogonal time-of-flight (OTOF) mass analyzer for mass analysis.
Postactivation—i.e. ion activation following the ETD reaction—is an important issue in ETD experiments. Swaney et al. (D. L. Swaney, G. C. McAlister, M. Wirtala, J. C. Scwartz, J. E. P. Syka, and J. Coon, Anal. Chem. 79, 477 (2007).) have shown that postactivation can substantially improve the fragmentation efficiency of ETD experiments. In ETD experiments an electron is transferred from the reagent ion to the analyte ion. In many cases, the energy from the resulting charge neutralization can fragment the analyte ion. However, in some cases a charge reduced nondissociated precursor ion is produced. In such cases additional energy is required to form fragment ions. The additional energy can be provided by accelerating the ions to a few eV of kinetic energy and then allowing the ions to collide with gas molecules in the trapping device. In a quadrupole trap this can be done by introducing a supplemental excitation waveform.
In the course of performing ion-ion reaction experiments such as ETD, it is often useful to trap a first reactant ion type in a first ion trap and a second reactant ion type in a second ion trap. The ions can then be allowed to mix and react for a well controlled, predetermined time.
When performing tandem mass spectrometry experiments in prior art traps, typically all analyte ions except for a single type of selected precursor ion are ejected from the trap. As a result, all ions except for the selected precursor are lost. Fragment ions may be formed from the selected precursor ion and these fragment ions may be further mass analyzed or fragmented, however, all other ions of potential interest originally stored in the trap are lost in the initial precursor isolation and are therefore unavailable for further analysis.
This is equally true of fragment ions when performing multiple step tandem mass spectrometry experiments. That is, if a precursor is selected, and if fragment ions are formed from the precursor, and then a single type of fragment ion is isolated for further fragmentation, then all the original ions except for the precursors will be lost and all the first generation fragment ions except for those isolated for further analysis will be lost.
As discussed below, the stacked well ion trap according to the present invention overcomes many of the limitations of prior art ion traps discussed above. The traps disclosed herein provides a unique combination of attributes making it especially suitable for use in the mass analysis of complex samples containing more than one type of analyte ion.